Gap junctions are aqueous channels permeable to ions and hydrophilic molecules of Mr < 1,000. Each gapjunctional channel is formed by head-to-head association of two connexons (connexin hexamers), one from each of the adjacent cells. The cochlea (responsible for mechano-transduction of sound waves into electrical impulses) has a highly-developed gap-junctional network that is essential for hearing. In most cells of the normal cochlea gap-junctional channels are heteromeric assemblies formed by the connexins Cx26 and Cx30. Infant deafness due to mutations of Cx26 is very common and our long-term objective is to elucidate at the molecular level the mechanisms by which mutations of connexins cause deafness. The properties of wild-type Cx26, Cx30 and heteromeric Cx26/Cx30 connexons, and the effects of mutations that cause deafness on the properties of heteromeric connexons that include wild-type connexins, are poorly understood. This is in part due to the fact that the few groups that have carried out functional experiments on Cx26 and Cx30 mutants work with complex systems, performing measurements that depend on the gapjunctional communication between two neighboring cells. This kind of experiments is essential to understand the effects of the mutations, but cannot fully address the molecular mechanism of the alterations. The specific aims of this proposal are: 1) to develop an expression/purification/reconstitution system that yields large amounts of functional wild-type and mutant Cx26 and Cx30, and 2) to test the usefulness of the purified connexon system to determine the functional properties of connexons formed by wild-type and mutant connexins. We will adapt the methodology that we developed recently for Cx43, which should allow us to obtain large amounts of functional Cx26 and Cx30, and connexin mutants. We will study the permeability properties of wild-type and mutant homomeric Cx26 and Cx30 connexons and heteromeric Cx26/Cx30 connexons formed by wild-type connexins as well as connexons containing Cx26 or Cx30 mutants. An integrative approach comparing the properties of gap- junctional channels and connexons will allow us to determine whether specific mutations alter gap-junctional communication at the level of the gap-junctional channel (e.g., docking between GJH) or connexon (e.g., non-permeable connexons).